Plk is the putative mammalian homologue of the Drosophila polo kinase and is implicated in regulating mitotic spindle formation and function. Plk kinase activity peaks during mitosis and Plk protein localizes at portions of the mitotic spindle and has been shown to associate with CHO1/klp, a kinesin-like motor protein. Using a variety of epitope tagged Plk truncation and deletion mutants we have identified a region of Plk that is both necessary and sufficient for nuclear localization of the protein. We have demonstrated that overexpression of wild type Plk transforms NIH3T3 cells in vitro and that such transformed cells form tumors in nude mice. Plk-induced transformation does not require a functional catalytic site or even full length Plk. Overexpression of a noncatalytic C-terminal region of Plk induces oncogenic focus formation. Fluorescence microscopy indicates that cells transformed by wild type or truncated Plk are highly aneuploid and frequently form abnormal mitotic spindles. Aberrant chromosome segregation caused by abnormal mitotic spindles may induce both aneuploidy and oncogenic transformation. Plk protein was found to be highly expressed in 16 of 57 human tumor cell lines and in several types of primary tumors. Deregulated expression of Plk may be involved in the origin or progression of some human cancers.